Main Cell Types Research Articles

The historical background of hereditary cystatin C amyloid angiopathy: Genealogical, pathological, and clinical manifestations.

Hereditary cystatin C amyloid angiopathy (HCCAA) is an Icelandic disease that belongs to a disease class called cerebral amyloid angiopathy, a group of heterogenous diseases presenting with aggregation of amyloid complexes and deposition predominantly in the central nervous system. HCCAA is dominantly inherited, caused by L68Q mutation in the cystatin C gene, leading to aggregation of the cystatin C protein. HCCAA is a very progressive and severe disease, with widespread cerebral and parenchymal cystatin C and collagen IV deposition within the central nervous system (CNS) but also in other organs in the body, for example, in the skin. Most L68Q carriers have clinical symptoms characterized by recurrent hemorrhages and dementia, between the age of 20-30 years. If the carriers survive the first hemorrhage, the frequency and severity of the hemorrhages tend to increase, resulting in death at average of 30 years with mean number of major hemorrhages ranging from 3.2 to 3.9 over a 5-year average life span. The pathogenesis of the disease in carriers is very similar in the CNS and in the skin based on autopsy studies, thus skin biopsies can be used to monitor the progression of the disease by quantifying the cystatin C immunoreactivity. The cystatin C deposition always colocalizes with collagen IV and fibroblasts in the skin are found to be the main cell type responsible for the deposition of both proteins. No therapy is available for this devastating disease.

Photo-Cross-linked Gelatin Methacryloyl Hydrogels Enable the Growth of Primary Human Endometrial Stromal Cells and Epithelial Gland Organoids.

In vitro three-dimensional (3D) models are better able to replicate the complexity of real organs and tissues than 2D monolayer models. The human endometrium, the inner lining of the uterus, undergoes complex changes during the menstrual cycle and pregnancy. These changes occur in response to steroid hormone fluctuations and elicit crosstalk between the epithelial and stromal cell compartments, and dysregulations are associated with a variety of pregnancy disorders. Despite the importance of the endometrium in embryo implantation and pregnancy establishment, there is a lack of in vitro models that recapitulate tissue structure and function and as such a growing demand for extracellular matrix hydrogels that can support 3D cell culture. To be physiologically relevant, an in vitro model requires mechanical and biochemical cues that mimic those of the ECM found in the native tissue. We report a semisynthetic gelatin methacryloyl (GelMA) hydrogel that combines the bioactive properties of natural hydrogels with the tunability and reproducibility of synthetic materials. We then describe a simple protocol whereby cells can quickly be encapsulated in GelMA hydrogels. We investigate the suitability of GelMA hydrogel to support the development of an endometrial model by culturing the main endometrial cell types: stromal cells and epithelial cells. We also demonstrate how the mechanical and biochemical properties of GelMA hydrogels can be tailored to support the growth and maintenance of epithelial gland organoids that emerge upon 3D culturing of primary endometrial epithelial progenitor cells in a defined chemical medium. We furthermore demonstrate the ability of GelMA hydrogels to support the viability of stromal cells and their function measured by monitoring decidualization in response to steroid hormones. This study describes the first steps toward the development of a hydrogel matrix-based model that recapitulates the structure and function of the native endometrium and could support applications in understanding reproductive failure.

Single-cell RNA sequencing uncovers cellular heterogeneity and the progression of heterotopic ossification of the elbow.

Heterotopic ossification of the elbow (HOE) is a complicated pathologic process characterized by extra bone formation in the elbow. Bone formation is a complex developmental process involving the differentiation of mesenchymal stem cells into osteoblasts. The aim of this study was to explore the cellular origin and progression of HOE by single-cell RNA sequencing. We identified 13 clusters of cells in HOE and further analyzed the subclusters for 4 of the main cell types. Six subclusters of osteoblasts, nine subclusters of chondrocytes, six subclusters of fibroblasts, and five subclusters of mononuclear phagocytes (MPs) were identified and analyzed. The new findings on osterix (OSX) and SOX9 expression in osteoblast subclusters and chondrocyte subclusters indicate that HOE is mediated through endochondral ossification. Further identification of the corresponding signature gene sets of distinct subclusters indicated that subclusters of osteoblasts_3, osteoblasts_4, osteoblasts_5, and osteoblasts_6 are relatively more mature during the osteoblastic progression of HOE. The trajectory analysis of the osteoblasts demonstrated that some genes were gradually downregulated, such as CRYAB, CCL3, SFRP4, WIF1, and IGFBP3, while other critical genes were upregulated, such as VCAN, IGFBP4, FSTL1, POSTN, MDK, THBS2, and ALPL, suggesting that these factors may participate in HOE progression. Cell-cell communication networks revealed extensive molecular interactions among the 13 HOE clusters. Ligand-receptor pairs for IL6, COL24A1, COL22A1, VWF, FZD6, FGF2, and NOTCH1 were identified, suggesting that multiple signaling pathways may be involved in HOE. In conclusion, this study provided the cellular atlas for HOE. We have established a greater extent of the heterogeneity of HOE cells than previously known through transcriptomic analysis at the single-cell level. We have observed gradual patterns of signature gene expression during the differentiation and maturation progression of osteoblasts from stem cells in HOE with higher resolution. The cell heterogeneity of HOE deserves further investigation to pave the way for identification of potential targets for HOE early diagnosis and therapeutic treatment.

Modern approaches to the creation of 2D and 3D cellular models for the study of lung fibrosis

Lung fibrosis is a consequence of many influences leading to damage to lung tissue and the development of subsequent inflammation. Fibrosis is an overgrowth of connective tissue, which can lead to a violation of the architectonics of the lungs and reduce their functionality up to a fatal outcome. At the same time, the mechanisms underlying fibrogenesis are currently insufficiently studied. In this regard, the task of studying them does not lose its relevance, and its solution requires the development of models of lung fibrosis that can reflect all the key processes of fibrogenesis.The in vivo model using animals has multiple undeniable advantages, but at the same time it has strict ethical limitations and does not reflect all the mechanisms of lung fibrosis inherent in the human body. At the same time, in vitro research, scientists can afford to use biomaterials not only of animals, but also of humans, and build cellular systems based on them — from 2D to 3D models. Modeling of pulmonary fibrosis is mainly based on the use of the main types of cells involved in the development of pulmonary fibrosis, such as myofibroblasts, fibroblasts, alveolocytes and others. Some models are also based on a specific fibrosis-associated extracellular matrix and further study of the interaction of cells with each other and with the matrix. It should be borne in mind that different models display individual nuances of the native processes of lung fibrogenesis, which requires the research community to use a wide range of models. Taking into account the multifactorial pathogenesis of pulmonary fibrosis, it is important to understand the totality of the processes taking place in order to obtain the completeness of the real picture, close to the picture in vivo, and therefore the multicomponence of models is important. This review focuses on the analysis of various models of lung fibrosis in vitro in two-dimensional and three-dimensional systems, shows approaches to their creation, key differences, main advantages and disadvantages of models, both particular and general.

Computational approaches identify a transcriptomic fingerprint of drug-induced structural cardiotoxicity

Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to improve the mechanistic understanding of SCT. First, we combined machine learning methods with a modified calcium transient assay in human-induced pluripotent stem cell-derived cardiomyocytes to identify nine parameters that could predict SCT. Next, we applied transcriptomic profiling to human cardiac microtissues exposed to structural and non-structural cardiotoxins. Fifty-two genes expressed across the three main cell types in the heart (cardiomyocytes, endothelial cells, and fibroblasts) were prioritised in differential expression and network clustering analyses and could be linked to known mechanisms of SCT. This transcriptomic fingerprint may prove useful for generating strategies to mitigate SCT risk in early drug discovery.Graphical

All GLP-1 Agonists Should, Theoretically, Cure Alzheimer's Dementia but Dulaglutide Might Be More Effective Than the Others.

Addressing the dysfunctions of all brain cell types in Alzheimer's disease (AD) should cure the dementia, an objective that might be achieved by GLP-1 agonist drugs, because receptors for GLP-1 are present in all of the main brain cell types, i.e., neurons, oligodendroglia, astroglia, microglia, endothelial cells and pericytes. This article describes the benefits provided to all of those brain cell types by GLP-1 agonist drugs. The article uses studies in humans, not rodents, to describe the effect of GLP-1 agonists upon cognition, because rodents' brains differ from those of humans in so many ways that results from rodent studies may not be totally transferable to humans. Commercially available GLP-1 agonists have mostly shown either positive effects upon cognition or no effects. One important reason for no effects is a reduced rate of entering brain parenchyma. Dulaglutide has the greatest entry to brain, at 61.8%, among the available GLP-1 agonists, and seems to offer the best likelihood for cure of AD. Although there is only one study of cognition that used dulaglutide, it was randomized, placebo controlled, and very large; it involved 8828 participants and showed significant benefit to cognition. A clinical trial to test the hypothesis that dulaglutide may cure AD should have, as its primary outcome, a 30% greater cure rate of AD by dulaglutide than that achieved by an equipoise arm of, e.g., lithium plus memantine.

Mapping the Spatial Dynamics of the CD4+ T Cell Spectrum in Classical Hodgkin Lymphoma

As around 25% to 30% of classical Hodgkin lymphoma (cHL) patients with advanced stages do not respond to standard therapies, the tumor microenvironment of cHL is one avenue that may be explored with the aim of improving risk stratification. CD4+ T cells are thought to be one of the main cell types in the tumor microenvironment. However, few immune signatures have been studied, and many of these lack related spatial data. Thus, our aim is to spatially resolve the CD4+ T cell subtypes that influence cHL outcome, depicting new immune signatures or transcriptional patterns that are in crosstalk with the tumor cells. This study was conducted using the NanoString GeoMx digital spatial profiling technology, based on the selection of distinct functional areas of patients’ tissues followed by gene-expression profiling. The goals were to assess the differences in CD4+ T cell populations between tumor-rich and immune-predominant areas defined by different CD30 and PD-L1 expression levels and seek correlations with clinical metadata. Our results depict a complex map of CD4+ T cells with different functions and differentiation states that are enriched at distinct locations, the flux of cytokines and chemokines that could be related to these, and the specific relationships with the clinical outcome.

Gliovascular transcriptional perturbations in Alzheimer’s disease reveal molecular mechanisms of blood brain barrier dysfunction

To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer’s disease, we performed single nucleus RNA sequencing in 24 Alzheimer’s disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands, SMAD3, upregulated in Alzheimer’s disease pericytes, has the highest number of ligands including VEGFA, downregulated in Alzheimer’s disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer’s disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here, we detect vast transcriptome changes in Alzheimer’s disease at the gliovascular-unit, prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions, and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer’s disease.

LMNA -Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-derived iPSC Differentiation Support Cell type and Lineage-specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells with Lamin A/C Haploinsufficiency.

LMNA -Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C ( LMNA ) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. Molecular mechanisms of disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA -Related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (4 Patient and 8 Control) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for Cardiac Progenitors to Cardiomyocytes (CM) and Epicardium-Derived Cells (EPDC). Data integration and comparative analyses of Patient and Control cells found cell type and lineage differentially expressed genes (DEG) with enrichment to support pathway dysregulation. Top DEG and enriched pathways included: 10 ZNF genes and RNA polymerase II transcription in Pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CM; LMNA and epigenetic regulation and DDIT4 and mTORC1 signaling in EPDC. Top DEG also included: XIST and other X-linked genes, six imprinted genes: SNRPN , PWAR6 , NDN , PEG10 , MEG3 , MEG8 , and enriched gene sets in metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

Single-cell RNA sequencing reveals the gene expression profile and cellular communication in human fetal heart development

The heart is the central organ of the circulatory system, and its proper development is vital to maintain human life. As fetal heart development is complex and poorly understood, we use single-cell RNA sequencing to profile the gene expression landscapes of human fetal hearts from the four-time points: 8, 10, 11, 17 gestational weeks (GW8, GW10, GW11, GW17), and identified 11 major types of cells: erythroid cells, fibroblasts, heart endothelial cells, ventricular cardiomyocytes, atrial cardiomyocytes, macrophage, DCs, smooth muscle, pericytes, neural cells, schwann cells. In addition, we identified a series of differentially expressed genes and signaling pathways in each cell type between different gestational weeks. Notably, we found that ANNEXIN, MIF, PTN, GRN signalling pathways were simple and fewer intercellular connections in GW8, however, they were significantly more complex and had more intercellular communication in GW10, GW11, and GW17. Notably, the interaction strength of OSM signalling pathways was gradually decreased during this period of time (from GW8 to GW17). Together, in this study, we presented a comprehensive and clear description of the differentiation processes of all the main cell types in the human fetal hearts, which may provide information and reference data for heart regeneration and heart disease treatment.

Explorations about the correlation between biological changes of meninges in periodontitis mice and cognitive impairment via single-cell RNA sequencing

Objective: To clarify the potential correlation between biological changes of meninges in periodontitis mice and cognitive impairment by analyzing the biological changes of meninges in periodontitis mice using single-cell RNA sequencing. Methods: Thirty C57BL/6 mice were divided into two groups by using random number table method (15 mice in each group). Mice in the control group were locally administered 2% carboxyl methyl cellulose (CMC) without Porphyromonas gingivalis (Pg) on both buccal sides. A mixture of Pg W83 and 2% CMC was applied on both buccal sides in the experimental group mice three times a week, lasting for 16 weeks in total. The absorption of alveolar bone, locomotor activity and cognitive function, the activation of microglia and astrocytes in the cortex were observed and assessed. The mRNA expression levels of Occludin in meninges and brain were detected in two groups. Single-cell RNA sequencing data of meninges were processed by uniform manifold approximation and projection (UMAP). Differential genes expressions of endothelial cells were processed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In addition, real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the expressions of transcription activating factor 3 (Atf3) and apolpoprotein L domain-containing 1 (Apold 1). Results: Methylene blue staining found the distances of buccal and palatal cement-enamel junction-alveolar bone crest in experimental mice [(185.60±17.60), (206.90±13.37) μm] increased significantly compared with the control group [(135.33±9.57), (163.05±14.98) μm] (t=5.02, P=0.002; t=4.37, P=0.005). Open field experiment showed the total distance and average speed of mice in the experimental group [(971.88±164.57) cm, (3.25±0.55) cm/s] were not statistically significant compared with the control group [(914.24±278.81) cm, (3.05±0.93) cm/s] (t=0.65, P=0.525; t=0.65, P=0.520). The recognition index of the experimental group [(48.02±16.92) %] was lower than the control group [(66.27±17.90) %] (t=2.40, P=0.027) by novel object recognition tests. Compared with the control group [(63.56±11.88) %], the alternation of experimental group [(50.99±14.17) %] was significantly decreased in Y maze tests (t=2.33, P=0.030). Immunohistochemistry results showed microglia and astrocytes were activated in the cortex of experimental mice. Compared with the control group (1.02±0.25, 1.04±0.31), the relative mRNA expressions of Occludin decreased significantly in the meninges and brain of periodontitis mice, respectively (0.61±0.10, 0.64±0.20) (t=3.47, P=0.010; t=2.66, P=0.024). By single-cell RNA sequencing, meninges cells were divided into 11 types, such as endothelial cells, fibroblasts, immune cells and so on. Endothelial cells were the main cell types in meninges [the control group: 26.47% (1 589/6 004), the experimental group: 26.26% (807/3 073)]. Compared with the control group [5.56% (334/6 004)], the percentage of granulocytes increased in the periodontitis mice [11.65% (358/3 073)]. Using clustering analysis to further focus on endothelial cells, GO enrichment analysis revealed differential genes were mainly related to angiogenesis, cell adhesion, apoptosis and so on. KEGG enrichment analysis revealed that differential genes were related to signaling pathways of interleukin-17, relaxin and so on. The relative mRNA expressions of Atf3 and Apold1 in meninges of periodontitis mice (0.42±0.24, 0.54±0.27) were significantly lower than the control group (1.03±0.26, 1.02±0.23) (t=3.88, P=0.005; t=3.02, P=0.017). Conclusions: The mice chronically infected with Pg W83 occurred memory impairment, neuroinflammation and changes of barrier function. In the meninges of periodontitis mice, there were infiltration of immune cells and down-regulation expressions of Atf3 and Apold1 by single-cell RNA sequencing. Meningeal immunity and changes of barrier function may play an important role in the cognitive impairment caused by periodontitis.

Single-nucleus analysis reveals microenvironment-specific neuron and glial cell enrichment in Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is a complicated neurodegenerative disease. Neuron-glial cell interactions are an important but not fully understood process in the progression of AD. We used bioinformatic methods to analyze single-nucleus RNA sequencing (snRNA-seq) data to investigate the cellular and molecular biological processes of AD.MethodsnRNA-seq data were downloaded from Gene Expression Omnibus (GEO) datasets and reprocessed to identify 240,804 single nuclei from healthy controls and patients with AD. The cellular composition of AD was further explored using Uniform Manifold Approximation and Projection (UMAP). Enrichment analysis for the functions of the DEGs was conducted and cell development trajectory analyses were used to reveal underlying cell fate decisions. iTALK was performed to identify ligand-receptor pairs among various cell types in the pathological ecological microenvironment of AD.ResultsSix cell types and multiple subclusters were identified based on the snRNA-seq data. A subcluster of neuron and glial cells co-expressing lncRNA-SNHG14, myocardin-related transcription factor A (MRTFA), and MRTFB was found to be more abundant in the AD group. This subcluster was enriched in mitogen-activated protein kinase (MAPK)-, immune-, and apoptosis-related pathways. Through molecular docking, we found that lncRNA-SNHG14 may bind MRTFA and MRTFB, resulting in an interaction between neurons and glial cells.ConclusionsThe findings of this study describe a regulatory relationship between lncRNA-SNHG14, MRTFA, and MRTFB in the six main cell types of AD. This relationship may contribute to microenvironment remodeling in AD and provide a theoretical basis for a more in-depth analysis of AD.

Identification of related-genes of T cells in lung tissue of chronic obstructive pulmonary disease based on bioinformatics and experimental validation

T cells are one of the main cell types shaping the immune microenvironment in chronic obstructive pulmonary disease (COPD). They persist andplay cytotoxic roles. The purpose of this study aimed to explore the potential related-genes of T cells in lung tissue of COPD. Chip data GSE38974 and single_celldata GSE196638 were downloaded from the GEO database. Difference analyses and WGCNA of GSE38974 were performed to identify DEGs and the modules most associated with the COPD phenotype. Various cell subsets were obtained by GSE196638, and DEGs of T cells were further identified. GO, GSEA and KEGG enrichment analyses were conducted to explore the biological functions and regulatory signaling pathways of the DEGs and DEGs of T cells. The intersection of the DEGs, module genes and DEGs of T cells was assessed to acquire related-genes of T cells. The mRNA and protein expression levels of related-genes ofT cells were verified in lung tissue of mouse with emphysema model. Based on GSE38974 difference analysis, 3811 DEGs were obtained. The results of WGCNA showed that the red module had the highest correlation coefficient with the COPD phenotype. GSE196638 analysis identified 124 DEGs of T cells. The GO, GSEAand KEGG enrichment analyses mainly identified genes involved in I-kappaB kinase/NF-kappaB signaling, receptor signaling pathway via STAT, regulationof CD4-positive cells, regulation of T-helper cell differentiation, chemokine signaling pathway, Toll-likereceptor signaling pathway, CD8-positive cells, alpha–beta T cell differentiation, MAPK signaling pathway and Th17 cell differentiation. The DEGs, genes of the red module and DEGs of T cells were overlapped to acquire FOXO1 and DDX17. The results of RT-qPCR and Western Blot indicate that the mRNA and protein expression levels of FOXO1 and DDX17 in lung tissue of emphysema mice were significantly higher compared with those in air-exposed mice. FOXO1 as well as DDX17 may be related-genesof T cells in lung tissue of patient with COPD, and their participation in the biological processes of different signaling pathways may inspire further COPD research.

#1914 Comprehensive single-cell sequencing and lipidomics identify diverse injury states and cholesterol dynamics of the PTs in AKI-to-CKD transition

Abstract Background and Aims The underlying cellular events and metabolic dysfunction of acute kidney injury (AKI) transformed to chronic kidney disease (CKD) is still largely unclear. Previous studies have suggested lipids and metabolites have a profound impact on acute and chronic inflammation. One of the main sites of renal lipid accumulation is the proximal tubule cells (PTs). Here we demonstrated the injury states and cholesterol dynamics of PTs and its potential function in AKI to CKD in unilateral ischemia-reperfusion injury (UIR) mice model. Method We employed single-cell combinatorial indexing lipidomics to analyze 15 mouse kidneys from a mouse model of failed repaired UIR at five time points. We identified cell types through unsupervised clustering analysis. Differential gene expression, enrichment analysis and main function scoring were applied to define functional heterogeneity and injury states of PTs. Next, pseudotime analysis of differentiation transitions was performed to create the diverse PT lineages. Finally, using SCENIC analysis, we identified the key transcription factors that drive the differentiation of PTs. Results We identified 13 main cell clusters and the dynamic changes in the process of AKI to CKD transition were analysed. A remarkable decrease in PTs after injury at day 1 followed with a recovery at day 3 post AKI were observed. Spatiotemporal profiling of the main cell types showed that the most injured areas in the acute stages of kidney injury are primarily concentrated in the outer stripe, specifically the region where the proximal straight tubules (PSTs) are located. In order to gain a higher resolution of the dynamics of PST subtypes during AKI to CKD, we performed sub-clustering analysis specifically on PSTs, partitioning them into 10 subclusters. Single-cell analysis identified maladaptive PSTs (PST-5) with the highest apoptosis and ferroptosis scores. Notably, pseudotime analysis revealed that PST-5 differentiated from PST-4, which has the highest lipid metabolism score. Indeed, among all the cells in the kidney, the PTs exhibited the most active metabolic reprogramming due to their differentiation and redifferentiation early after AKI. Lipid omics profiling reveals an abnormal elevation of the cholesterol metabolites 27-hydroxycholesterol (27-OH) and 25-hydroxycholesterol (25-OH) following AKI. Interestingly, the PST-4-specific expression of Cyp7b1 served as a key enzyme regulating the concentration of 27-OH. This gene has been found to be significantly absent after AKI. Moreover, the binding of 27HC, a natural ligand, to endogenous estrogen receptor alpha (ERα) leads to the upregulation of heme oxygenase 1 (Hmox1), thereby promoting ferroptosis. Finally, SCENIC analysis revealed that Cyp7b1-mediated accumulation of 27-HC is dependent on the transcription factor Atf3. Conclusion Our results indicate that Cyp7b1 deficiency confers ferroptosis, which are primarily dependent on the upregulation of cellular cholesterol homeostasis and 27-HC secretion, leading to the progression of AKI. These findings suggest that the Cyp7b1/Hmox1/Atf3/27-HC axis may serve as a potential therapeutic target for preventing the development of CKD.

Ontogenesis and variation of wood ‘parenchymatization’ in Cochlospermum vitifolium (Bixaceae)

Summary Axial parenchyma proliferation (non-lignified parenchyma) in wood has been characterized for only a few species. In the present study, we characterized the proliferation, ontogeny, and seasonal variation of wood axial parenchyma in Cochlospermum vitifolium (Bixaceae). This study included material from natural populations from different sites in Mexico. We analyzed the wood anatomy and development in stems of different ages, and monthly sampling of the main stem during the growing season. The results show that the wood anatomy of C. vitifolium is characterized by features associated with efficient water transport, such as wide vessels and low vessel frequency. The wood has a high storage and radial transport capacity. Axial parenchyma is abundant and constitutes the main cell type of the wood. Rays are wide and tall with a high starch content. Ontogenetic development of the wood showed that non-lignified axial parenchyma differentiates after secondary growth has been established. Parenchyma proliferation does not occur in an axially orderly fashion and the results suggest that there is a relationship between maximum proliferation and precipitation. We found high variation in wood parenchymatization between trees. The maximum parenchymatization and succulence was found in trees with larger stem diameter. The ecological and ontogenetic implications of wood parenchymatization are discussed.

ПРОЦЕССЫ РЕГЕНЕРАЦИИ КОСТНОЙ ТКАНИ ПРИ ПЕРЕЛОМАХ НИЖНЕЙ ЧЕЛЮСТИ

Item. The review of literature devoted to the actual problem of maxillofacial surgery — processes of bone tissue regeneration in mandibular fractures is presented. The purpose of the study — to study and analyze the materials of publications on the processes of bone tissue regeneration in mandibular fractures. Methodology. The publications of domestic and foreign authors devoted to the processes of bone tissue regeneration in mandibular fractures were studied. The publications were analyzed using scientific search library databases: PubMed Central, Google, Cyberleninka, eLibrary. Materials were selected by keywords. Full-text articles were included in the search. Results. In domestic and foreign literature, the processes of restoration of bone structures of the maxillofacial region as an organism's response to damage have been studied and described in detail. It has been established that two processes are simultaneously launched: resorption of damaged osteocytes and formation of osteoid tissue, one of which may be predominant at the moment. Conclusions. The article presents modern data on the key moments of bone tissue regeneration in the region of the mandible. All stages of the reparative process in bone tissue are described at the cellular level, and information about the main mechanisms, pathways and cell types involved in bone tissue regeneration is presented in detail, taking into account the stage of osteogenesis. Knowledge of the regularities of biological processes in the bone tissue regeneration zone allows scientific search and development of effective methods of treatment of mandibular fractures.

Abstract PO3-23-08: Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib

Abstract Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib. Marie Fongaard1, Leonard Schmiester2, Salim Ghannoum1, Pål Marius Bjørnstad1, Tatjana Bosnjak3, Signe Meltzer Kleivbo3, Knut Selsås4, Stephanie Beate Geisler5, Kamilla Fjermeros5, Sameer Bhargava5, Manouchehr Seyedzadeh6, Unn-Cathrin Buvarp5, Aino Katri Rosenskiold5, Nam Thi Nguyen5, Torben Lüders7, Diether Lambrechts8, Marianne Lyngra9, Arnoldo Frigessi2, Vessela Kristensen1,10, Jürgen Geisler5,10,*, Xavier Tekpli1,* 1) Department of Medical Genetics, Oslo University Hospital, Oslo, Norway 2) University of Oslo, Oslo Centre for Biostatistics and Epidemiology (OCBE), University of Oslo, Norway 3) Novartis, Medical Affairs, Oslo, Norway 4) Department of Breast and Endocrine Surgery, Akershus University Hospital, Oslo, Norway 5) Department of Oncology, Akershus University Hospital, Oslo, Norway 6) Department of Radiology, Akershus University Hospital, Oslo, Norway 7) Department of Clinical Molecular Biology (EPIGEN), Akershus University Hospital, Oslo, Norway 8) Laboratory for Translational Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium 9) Department of Pathology, Akershus University Hospital, Oslo, Norway 10) University of Oslo, Institute of Clinical Medicine, Faculty of Medicine, Oslo, Norway< * Jointly supervised the work, shared corresponding authors Background The recent introduction of CDK4/6 inhibitors is regarded as a major improvement in the field of breast cancer therapy. A growing body of evidence suggests that CDK4/6 inhibitors may impact on the recruitment of immune cells within the tumor microenvironment during therapy. Further, CDK4/6 inhibitors may potentially contribute to the extinction or survival of specific cancer cell clones through evolutionary pressure exerted on breast tumors. Clinical trial The NEOLETRIB-study is a multicenter, single-arm, open-label phase II clinical trial. Patients with locally advanced, LUM-A/LUM-B breast cancer, characterized by cT3-cT4 tumors and/or cN2-3 lymph node involvement, receive ribociclib (600 mg daily, 21 days on and 7 days off) in combination with letrozole (2.5 mg daily) for a duration of 6 months in the neoadjuvant setting. Patients with large cT2 tumors are also allowed to participate. Aim of the study Using single-cell analyses at different time points of treatment, we aim at studying the evolutionary trajectories of cancer and immune cells in 30 patients participating in the NEOLETRIB-trial. Methods Tumor biopsies taken before treatment (baseline) and during treatment (day 21 of ribociclib cycles 1 and 6, respectively), underwent single-cell RNA (scRNA), T cell receptor (TCR), and B cell receptor (BCR) sequencing. The sequencing data was first processed using cellRanger and Seurat . Results Initial clustering of the scRNA enabled the identification of the main cell types in breast tumors, which included T/NK cells, B cells, epithelial cells, cancer-associated fibroblasts, myeloid cells and endothelial cells. Focusing on epithelial cells, we were distinguished cancer cells from normal epithelial cells using the InferCNV algorithm. Further, by estimating the copy number events, we determined the genetic background of clones resistant or sensitive to combination therapies for each patient, revealing the dynamics of tumor heterogeneity evolution during treatment. Additionally, we examined CD4, CD8 and NK cell types, identified different cell states within these cell types and inferred pseudotime and immune cell type differentiation trajectories. ConclusionsWe identified the cell types present in tumor biopsies from the NEOLETRIB trial. We characterized the effects of therapies on the evolution of cancer and immune cells and gain insights into the factors influencing sensitivity and resistance to the combination of letrozole and ribociclib. Citation Format: Marie Fongård, Leonard Schmiester, Salim Ghannoum, Marius Bjørnstad, Tatjana Bosnjak, Signe Kleivbo, Knut Selsås, Stephanie Geisler, Kamilla Fjermeros, Sameer Bhargava, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Aino Vuoriluoto, Nam Thi Nguyen, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-08.

Abstract PO4-02-01: Characterization of immune and malignant cell evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen positive breast cancer

Abstract Background Aromatase inhibitors (AIs) are commonly used in the management of estrogen receptor-positive (ER+) breast cancer, as they effectively reduce estrogen levels which inhibits tumor growth and recurrence. In postmenopausal women with ER+ breast cancer, aromatase inhibitors are increasingly used in selected patients as neoadjuvant therapy to reduce the tumor burden before surgery and to improve overall treatment outcomes. Clinical trial The NEOLETEXE trial aimed to treat postmenopausal patients with locally advanced breast cancer. It was a neoadjuvant, randomized, open-label, intra-patient, cross-over, single-center phase II clinical trial. Most patients presented large T3/T4 tumors and/or N2/N3 involvement. Patients were randomly assigned to receive neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane (25 mg daily) for 3 months followed by a cross-over to the alternative AI for another 3 months followed by surgery. A total of 102 patients were enrolled in the NEOLETEXE trial. Aim of the study We aim at using single-cell analyses at different time points of treatment, to characterize evolutionary trajectories of cancer and immune cells in 23 patients from the NEOLETEXE trial. Methods Tumor biopsies taken before treatment (baseline) at crossover (after 3 months) and end of neoadjuvant therapy (after 6 months), were analyzed by single-cell RNA (scRNA), T cell receptor (TCR), and B cell receptor (BCR) sequencing. The data were first processed using cellRanger and the Seurat package. Results The main cell types found in tumors were identified through clustering of the scRNA data. We then focused on the epithelial cells to first distinguish between the normal and malignant epithelial cells using the InferCNV algorithm. For each patient, the analysis of inferred copy number events allowed to elucidate the genetic background of clones resistant or sensitive to aromatase inhibitors. This analysis unveiled the evolutionary dynamics of tumor heterogeneity during treatment. We next examined CD4, CD8, NK cells, and macrophages, using RNA velocity and diffusion pseudotime using CellRank and scVelo to delineate the differentiation trajectories of immune cell types. Conclusions We created a detailed map that provides a high-resolution view of the diverse cell types present in tumor biopsies from the NEOLETEXE trial. We characterize the impact of therapies on the evolutionary dynamics of both cancer and immune cells. Our analyzes sought insights into the underlying factors that contribute to the sensitivity and resistance to aromatase inhibitors. Citation Format: Leonard Schmiester, Salim Ghannoum, Marie Fongård, Marius Bjørnstad, Knut Selsås, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Characterization of immune and malignant cell evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-02-01.

Modelo de cocultivo 3D in vitro de células endoteliales y vasculares de músculo liso humanas para el estudio del remodelado vascular patológico

Pathological vascular remodeling of the vessel wall refers to the structural and functional changes of the vessel wall that occur in response to injury that eventually leads to cardiovascular disease. The vessel wall is composed of two main types of cells, endothelial cells and vascular smooth muscle cells, whose communication is crucial in both the development of the vasculature and the homeostasis of mature vessels. Changes in the dialogue between endothelial cells and vascular smooth muscle cells are associated with various pathological states that triggers remodeling of the vascular wall. For many years, considerable efforts have been made to develop effective diagnoses and treatments for these pathologies by studying their mechanisms in both in vitro and in vivo models. Compared to animal models, in vitro models can provide great opportunities to obtain data in a more homogeneous, economical and massive way, providing an overview of the signaling pathways responsible for these pathologies. The implementation of three-dimensional in vitro co-culture models for the study of other pathologies has been postulated as a potentially applicable methodology, which determines the importance of its application in studies of cardiovascular diseases. In this article we present a method for culturing human endothelial cells and vascular smooth muscle cells, grown under non-adherent conditions, that generate three-dimensional spheroidal structures with greater physiological equivalence to in vivo conditions. This in vitro modeling could be used as a study tool to identify cellular and molecular mechanisms involved in the pathological processes underlying vascular remodeling.

Host guanylate binding proteins participate in parasite control during Leishmania infection

Abstract Cutaneous leishmaniasis (CL) is a debilitating neglected tropical disease causing lesions ranging from self-healing to permanent disfigurations. A predominant Th1 response relying on IFNγ production leads to parasite control during CL. Although IFNγ is mainly responsible for activating macrophages to produce NO leading to parasite control, IFNγ can activate other downstream pathways involved in cell autonomous immunity including the activation of guanylate binding proteins (GBPs), a class of interferon-inducible GTPases. GBPs play a role in host defense against some intracellular pathogens, but minimal work has been done to understand the role of GBPs during Leishmania infection. Utilizing RNA-Seq we found several GBPs are upregulated in lesions during L. major infection in mice. Single-cell RNA-Sequencing revealed macrophages as the main cell type expressing Gbps, including Gbp2, Gbp5, and Gbp7 during infection. Additionally, Gbp expression was not unique to L. major infection; both L. amazonensis and L. mexicana infection in vivo resulted in elevated Gbp expression. Preliminary in vitro experiments show macrophages from GBPChr3 KO mice harbor increased numbers of parasites compared to control macrophages with and without IFNγ treatment. Consistent with in vitro findings, infected GBPChr3 KO mice exhibit increased pathology compared to control mice. Together these data suggest GBPs participate in the defense against Leishmania parasites.